biology Article The Impact of Host Genotype, Intestinal Sites and Probiotics Supplementation on the Gut Microbiota Composition and Diversity in Sheep Xiaoqi Wang 1,2,3, Zhichao Zhang 3, Xiaoping Wang 3, Qi Bao 3, Rujing Wang 1,2,* and Ziyuan Duan 3,* 1 Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, China; [email protected] 2 Science Island Branch of Graduate School, University of Science and Technology of China, Hefei 230026, China 3 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; [email protected] (Z.Z.); [email protected] (X.W.); [email protected] (Q.B.) * Correspondence: [email protected] (R.W.); [email protected] (Z.D.); Tel.: +86-551-6559-2968 (R.W.); +86-10-6480-3631 (Z.D.) Simple Summary: Given the fact that sheep serve as an increased meat product consumption in China, as well as a suitable model for fat deposition, their digestive tract microbiota has drawn growing attention. Our study depicted the gut microbiota community composition and diversity of sheep with varied genotypes but shared geography, with different sampling intestinal sites and probiotics supplementation time. The results indicated the great difference of the gut microbiota in samples from different sheep breeds, various intestinal sites, and different probiotics feeding times. Citation: Wang, X.; Zhang, Z.; Additionally, all results implied the dominance of the host factor in shaping unique microbiota under Wang, X.; Bao, Q.; Wang, R.; Duan, Z. a certain environment, the greatest similarity of colonic and fecal microbiota, and the oral probiotic The Impact of Host Genotype, effectiveness for a given period of time for sheep. Intestinal Sites and Probiotics Supplementation on the Gut Abstract: Three sampling strategies with a 16s rRNA high-throughput sequencing and gene ex- Microbiota Composition and pression assay (by RT-PCR) were designed, to better understand the host and probiotics effect on Diversity in Sheep. Biology 2021, 10, gut microbiota in sheep. Sampling: (1) colon contents and back-fat tissues from small-tailed Han 769. https://doi.org/10.3390/ sheep (SHS), big-tailed Hulun Buir sheep (BHBS), and short-tailed Steppe sheep (SHBS) (n = 12, biology10080769 14, 12); (2) jejunum, cecum and colon contents, and feces from Tan sheep (TS, n = 6); (3) feces from TS at 4 time points (nonfeeding, 30 and 60 feeding days, and stop feeding 30 days) with probiotics Academic Editor: Ger Rijkers supplementation (n = 7). The results indicated SHS had the highest Firmicutes abundance, the thinnest back-fat, and the lowest expression of C/EBPb, C/EBPd, ATGL, CFD, and SREBP1. Some bacteria Received: 6 July 2021 Accepted: 10 August 2021 orders and families could be potential biomarkers for sheep breeds with a distinct distribution of Published: 12 August 2021 bacterial abundance, implying the host genotype is predominant in shaping unique microbiota under a shared environment. The microbiota diversity and Bifidobacterial populations significantly changed Publisher’s Note: MDPI stays neutral after 60 days of feeding but restored to its initial state, with mostly colonies, after 30 days ceased. The with regard to jurisdictional claims in microbiota composition was greatly different between the small and large intestines, but somewhat published maps and institutional affil- different between the large intestine and feces; feces may be reliable for studying large intestinal iations. microbiota in ruminants. Keywords: gut microbiota; composition; intestinal sites; sheep breeds; Bifidobacterium; feces Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article 1. Introduction distributed under the terms and There have already been many researches and achievements demonstrating the im- conditions of the Creative Commons portance of gastrointestinal tract bacteria, which plays an important role in the growth, Attribution (CC BY) license (https:// metabolism, and immunity of hosts [1,2]. Especially the intestinal microbiota composition creativecommons.org/licenses/by/ and stability, containing a temporal and spatial distribution status of bacteria [3], have a 4.0/). Biology 2021, 10, 769. https://doi.org/10.3390/biology10080769 https://www.mdpi.com/journal/biology Biology 2021, 10, 769 2 of 16 close relation with the host’s health and production performance of economically important animals [4]. It is then required to understand the intestinal microbial composition and its perturbations among economically important species, due to the production of enzymes for the digestion of cellulose and other plant polysaccharides, as well as the synthesis of certain vitamins [4,5]. Recent studies reported that diet, environment, and host genetic factors were im- portant in maintaining or affecting intestinal microbiota composition [6–8]. Nevertheless, with the object and region of study expanding, the extent of influence on the microbiota composition and diversity remains controversial. A research, based on 709 cattle, indicated the effect of host genetics on rumen microbiota [9], but less research focused on sheep. In this study, we employed four types of sheep breeds, which are the small-tailed Han sheep (SHS), the big-tailed Hulun Buir sheep (BHBS), the short-tailed Steppe sheep (also named the small-tailed Hulun Buir sheep, SHBS), and the Tan sheep (TS) to explore how the host genetic background and dietary supplement impact the intestinal microbiota composition and stability in sheep (Table S1). These four types of sheep breeds originated from different types of wild ancestors [10–12], but all belonged to the Mongolian sheep group, according to their development and breed formation. The genetic differentiation relationship of SHS, BHBS, SHBS, and TS, analyzed by microsatellite loci, also supports that the 4 sheep breeds originated from Mongolian sheep, which is consistent with the history of breed formation [13]. Owing to similar appearance and totally different tail shape, BHBS and SHBS are the great model to study fat deposition [10], while SHS is one of the most widely distributed sheep breeds in China, due to the strong reproductive capacity, hereditary stability, and better adaptation [11]. To this day, BHBS, SHBS, and TS are the main meat sheep in Hulun Buir region and Ningxia Hui Autonomous Region in China. Previous studies have reported gastrointestinal microbial community and diversity differences along the gastrointestinal tract of sheep [14]. Although the differences between small and the large intestinal microbiota are parallel, the great controversy was whether feces could represent large intestinal contents on the intestinal microbiota study [15,16]. Here, with the expectation of clarifying this issue, we selected TS to compare the similarities and differences of intestinal microbiota composition among different intestinal sites. According to the point of ecological view, diet could select microbial communities within the gut ecosystem [17], but the effect of dietary components on the stability of the gut microbiota has been poorly addressed [18]. Antibiotics, as a dietary supplemental component, would put on a prominent selective pressure on the gut microbiota and induce the most drastic perturbations as members of the gut microbiota [19]. Current studies on probiotics, modulating the intestinal microbiota via oral administration in monogastric animals [20] and rumen microbiota of ruminants [21,22], have been reported many times, but the effect and acting time of probiotics remain highly uncertain after by-passing the rumen of ruminants. It has been reported that Bifidobacterium supplementation can affect lipid metabolism or decrease pathogenic microbes [23], and thereby affects intestinal microstability, hence the correct acting time and effectiveness of Bifidobacterium would play a meaningful role in the sheep gut ecosystem and its productive performance. With ongoing studies on gastrointestinal tract microbiota, the above are common issues in sheep production and research. Therefore, we suggested the hypothesis: (1) host genetics influence intestinal microbial features that could relate to host productive performance of sheep in the same habitat; (2) fecal samples could substitute for large intestinal contents, to a certain extent, when characterizing large intestinal microbiota; (3) there has been a specific time for the onset and duration of oral Bifidobacterium in sheep. In this study, we have attempted to verify the hypothesis by 3 sampling strategies and provided reliable reference for sheep production and research. Biology 2021, 10, 769 3 of 16 2. Materials and Methods 2.1. Animals and Sampling 1. Samples from 3 different sheep breeds, with each breed belonging to one group. Colon contents and back-fat samples were collected from SHS (n = 12), BHBS (n = 14), and SHBS (n = 12), which were raised concurrently in the same manage condition, with the same forage in the Breeding Sheep Farm of state-owned Bayan Farm (Inner Mongolia Autonomous Region, China) from 4-month to 8-month age. The final body weights (FBW) ± SD of SHS, BHBS, and SHBS were 40.38 ± 6.00 kg, 38.90 ± 5.37 kg, and 37.47 ± 5.77 kg, respectively. 2. Samples from different intestinal sites. TS (female, 4-month-old, n = 6) were purchased from Ningxia livestock farm (Ningxia Hui Autonomous Region, China) and fed with commercial diets and raised in the same condition. After feeding 3 months, the intestinal content samples were collected from jejunum,